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In animals, double-stranded short interfering RNA (siRNA) and single-stranded microRNA (miRNA) regulate gene expression by targeting homologous mRNA for cleavage or by interfering with their translation, respectively . siRNAs are processed from injected or transgene-derived, long, perfect double-stranded RNA (dsRNA), while miRNAs are processed from short, imperfect dsRNA precursors transcribed from endogenous intergenic regions . In plants, both siRNAs and miRNAs activate cleavage of homologous RNA targets , but little is known about the genes controlling their production or action. The SGS2/SDE1 protein contributes to produce transgene siRNA , while DCL1 and HEN1 contribute to endogenous miRNA accumulation . Here, we show that: i) SGS2, SGS3 , AGO1 , and HEN1 contribute to produce transgene siRNA involved in sense posttranscriptional gene silencing (S-PTGS); ii) HEN1, but not SGS2, SGS3, or AGO1, contributes to the accumulation of the endogenous miR171 miRNA and to the cleavage of Scarecrow target mRNA by miR171 ; iii) SGS2, SGS3, AGO1, and HEN1 contribute to resistance against cucumber mosaic virus , but not to siRNA and IR-PTGS triggered by hairpin transgenes directly producing perfect dsRNA ; and iv) the actions of HEN1 in miRNA/development and siRNA/S-PTGS can be uncoupled by single-point mutations at different positions in the protein. [Copyright &y& Elsevier]

Cellular RNA-dependent RNA polymerases (RDRs) are fundamental components of RNA silencing in plants and many other eukaryotes. In Arabidopsis thaliana genetic studies have demonstrated that RDR2 and RDR6 are involved in the synthesis of double stranded RNA (dsRNA) from single stranded RNA (ssRNA) targeted by RNA silencing. The dsRNA is subsequently cleaved by the ribonuclease DICER-like into secondary small interfering RNAs (siRNAs) that reinforce and/or maintain the silenced state of the target RNA. Models of RNA silencing propose that RDRs could use primer-independent and primer-dependent initiation to generate dsRNA from a transcript targeted by primary siRNA or microRNA (miRNA). However, the biochemical activities of RDR proteins are still partly understood. Here, we obtained active recombinant RDR2 and RDR6 in a purified form. We demonstrate that RDR2 and RDR6 have primer-independent and primer-dependent RNA polymerase activities with different efficiencies. We further show that RDR2 and RDR6 can initiate dsRNA synthesis either by elongation of 21- to 24- nucleotides RNAs hybridized to complementary RNA template or by elongation of self-primed RNA template. These findings provide new insights into our understanding of the molecular mechanisms of RNA silencing in plants.